Our laboratory has a long-standing interest in immunochemical methods for cell separation, and in developing engineered macromolecular reagents for use in studying relatively low affinity interactions (such as those of MHC molecules with peptides and T cell receptors). We currently study engineered recombinant single chain Class I MHC molecules, with respect to their cell biology, immune function, and potential use as vaccine components. During this year, we have further characterized our original construct- the recombinant soluble single chain H-2Dd Class I MHC molecule, sscH-2Dd. We have found that sscH-2Dd as secreted contains a complement of bound endogenous peptides similar to those found in wild type two chain molecules. This provides further evidence that recombinant sscH-2D has a native conformation. We have developed a procedure for affinity purification of the single chain molecules, and for their uniform loading with a single antigenic peptide. Such uniform loading makes it possible to investigate their immunogenicity. Class I molecules function in nature as cell surface molecules. Dr. Lee has engineered a recombinant gene for a cell-surface bound single chain Class I MHC molecule. Stable L cell transfectants with this gene express the single chain molecule on their surface. The cells can bind an antigenic peptide from HIV gpl20, and are functional in antigen presentation to T cells. The single chain molecule can also be stably expressed by cells that lack endogenous beta2-microglobulin. This shows that the single chain molecule uses its own covalently linked beta2m, rather than endogenous beta2m. Finally, we have constructed recombinant genes for three additional single chain Class I MHC molecules:H-2Dd with a covalently linked antigenc peptide, H-2Ld, and a human Class I MHC molecule:HLA/A2.